Circulation Research, vol 112, 2013

Sereti et al identify cell-surface protein Abcg2 as a promoter of both proliferation and symmetric cell division in cardiac progenitors.

It is currently believed that to maintain homeostasis, cardiac progenitor cells divide asymmetrically, giving rise to one cell that differentiates and another that replaces the progenitor. After an injury, however, it is thought that the progenitor cell population is depleted as these cells differentiate into cardiac myocytes, and that replenishment of the pro genitors occurs by symmetrical division. Understanding the molecular mechanisms that drive cardiac progenitor cell division might therefore be useful in developing potential cell-based therapies for heart repair. The cell surface protein Abcg2, which is expressed on cardiac progenitors, was previously found to regulate proliferation in these cells. Now Sereti et al have discovered that this protein not only promotes cell-cycle progression, but also symmetric division. Progenitor cells that lacked Abcg2 tended to arrest before DNA replication—at G0 and G1 stages of the cell cycles—while those that did divide tended to do so asymmetrically. Thus, the authors suggest that regulating Abcg2 levels after injury could maximize the regenerative potential of the heart.

It is well known that nitric oxide (NO) protects blood vessels during ischemia. And it has also been reported that the calcium-binding protein S100A1, expressed in endothelial cells, increases NO synthesis via eNOS. Furthermore, S100A1 has been found to be crucial for endothelial function. Most et al now bring these separate pieces of evidence together and show that ischemic tissue samples taken from both patients with chronic critical limb ischemia and mice with induced limb ischemia display a near-complete loss of S100A1 expression, which is associated with a decrease in NO. The team created S100A1 knockout (SKO) mice and showed that these animals have impaired reperfusion and neovascularization after ischemia. However NO injections restored the angiogenic potential in these mice. The team also showed that S100A1 directly binds and activates eNOS and also indirectly activates the enzyme by suppressing the activity of an eNOS inhibitor. Altogether, these results show that S100A1 is a critical regulator of eNOS and that it could be a therapeutically important protein for the treatment of ischemic vascular disease.

Vrtovec et al report the results of a 5-year follow-up study of dilated cardiomyopathy (DCM) showing that patients who received intracoronary bone marrow transplants have better exercise capacity, heart function and better chance of survival compared with patients that did not receive the treatment. The 55 randomly selected DCM patients who received the bone marrow generally had higher left ventricle ejection fraction, could walk further, had lower levels of NT-proBNP—a protein marker of heart failure—in their blood, and fewer of them died. Interestingly, symptomatic improvement correlated with the extent to which the bone marrow cells integrated into the myocardium—which was assessed by radioactive tracing on the day of the transplantation. The improvements generally tended to be most significant within the first few years of the procedure. Ejection fraction, for example, showed a dramatic difference between test subjects and controls after one year, which persisted for a further two years, but then started to decline. This suggests that repeating the treatment regimen every few years, might further improve outcomes in DCM patients, say the authors.

Ibarra et al reveal how calcium signals get to cardiac myocyte nuclei without interference from the cytoplasm.

Calcium signaling in the nucleus regulates several cell functions including, chromatin condensation, nuclear transport and the activation of transcription factors. However, in cardiac myocytes, where the calcium signals in the cytosol are in constant flux, it is unclear how nuclear calcium signals are separated from the cytosolic noise. Stimulation of the cell surface receptor, insulin-like growth factor 1 receptor (IGF-1R), has been known to activate nuclear calcium signaling in cardiac myocytes, but how this signal is transmitted has remained a mystery. Now Ibarra et al show that IGF-1R is localized to invaginations of the plasma membrane or T-tubules that reach right up to the nucleus. They found that components of the signaling cascade, downstream of IGF-1R, including an associated G-protein, inositol 1,4,5 triphosphate, and phospholipase C, are also localized to the nuclear region. Furthermore, IGF-1R adopted a similar location and function in embryonic and neonatal cardiac myocytes, where T-tubules are thought to be underdeveloped. The authors suggest that the newly discovered nuclear signaling function of T-tubules might be of clinical relevance as dysfunctional T-tubules are associated with heart disease and myocardial ischemic injury.

Critical limb is chemia (CLI) is an advanced form of peripheral artery disease that can cause extensive tissue injury and cell death. In severe cases, amputation is the only option. To prevent the need for such drastic treatments, researchers are investigating the use of autologous bone-marrow pro-angiogenic cells (PACs) for therapy. PAC function is impaired in CLI patients, however, so Spinetti et al wanted to know why. They found that 2 microRNAs, miR-15a and miR-16, were over-expressed in PACs and were associated with impaired survival and migration of these cells. Over-expressing miR-15a and miR-16 in healthy PACs increased their rate of apoptosis and decreased migration, while suppressing expression of the miRs in patient PACs, improved cell migration. Furthermore, PACs in which miR-15a and -16 expression was repressed, improved post-ischemic blood flow and vessel density when injected into mice with limb ischemia. The authors suggest that not only could the miRs serve as biomarkers of the disease, but that repression of miR-15a and -16 might improve PAC-based therapies in patients with CLI.

Chronic pulmonary arterial hypertension (PAH) is not, as the name might suggest, a problem restricted to the arteries of the lungs. It is also associated with decreased functioning of the right ventricle of the heart, which can ultimately lead to heart failure and death. Endothelin receptor antagonists are currently used for the treatment of PAH, but Nagendran et al suggest that despite their favorable profile, these drugs might have detrimental effects. The drugs lower blood pressure by blocking the activity of a vasoconstrictor called Endothelin-1, which is upregulated in the pulmonary arteries of patients with PAH. However, the authors show that both Endothelin-1 and the Endothelin receptor are also upregulated in the right ventricle of these patients, and in rats with PAH. In the heart, Endothelin-1 might be part of an important compensatory response to maintain contractility, the authors suggest. Indeed, ex vivo treatment of PAH model rat hearts with an Endothelin receptor antagonist reduced contractility. On the basis of these observations, they caution that direct cardiac effects of endothelin receptor antagonists should be taken into account when considering the use of these drugs to treat PAH patients.

During heart failure, β-adrenergic stimulation of the heart is increased to aid in contraction, providing temporary relief to weakened heart muscles. In the long-term however, this stimulation causes cardiac myocytes to die. To prevent this damage β-adrenergic blockers are often used to treat heart failure patients. When cardiac myocytes die, they are replaced, albeit slowly, by cardiac progenitor cells. However, it is unclear how exactly these progenitors are affected by β-blockers, or indeed, by β-adrenergic stimulation. Now, Khan et al report that β-adrenergic stimulation induces mouse cardiac progenitor cells to proliferate. But, just like adult cardiac myocytes, the progenitor cells underwent apoptosis as soon as they were induced to differentiate. The investigators found that progenitor cells expressed only one type of β-adrenergic receptor, β1-AR, but upon differentiation, they started to express a second, β2-AR—also found in mature cardiac myocytes. And because these 2 receptors convey separate effects, the authors suggest that a bipartite approach, involving β1-AR activation and β2-AR blockade, could be more effective in treating heart failure.

Much like Khan and colleagues, Zhang et al were interested in the effects of β-adrenergic stimulation and β-blockers on the heart. They knew that long-term β-adrenergic stimulationduring heart failure causes heart cells to die. They also knew that β-blockers prevent this cell death. However, the molecular details of these processes were unclear. β-Adrenergic–induced cell death is thought to be mediated by the stimulation of protein kinase A (PKA), but the effects of β-adrenergic stimulation could also be PKA-independent. To differentiate between PKA and non-PKA effects, the team created transgenic mice expressing a PKA inhibitor. They found that β-adrenergic stimulation in these mice prevented cardiac myocyte death and that the level of protection offered by PKA inhibition was similar to that provided by β-blockers. There was one important distinction, however. The hearts of the transgenic mice when stimulated also exhibited marked ERK activation, which offered further protection to cardiac myocytes. Following myocardial infarction, these PKA-inhibited mice also showed greater improvement in cardiac function than wild-type mice treated with β-blockers. The authors suggest that for treating heart failure patients, PKA-inhibitors may be more effective than β-blockers.

Cardiovascular complications, such as atherosclerosis and capillary damage are common in diabetes and can have far reaching clinical consequences. In mouse models such capillary damage can affect even the bone marrow. Worse still, in the bone marrow, the failing microvasculature, which feeds the stem cell niche, could reduce the production of progenitors, including pro-angiogenic cells. While it has been shown that circulating progenitor cell levels are suppressed in people with diabetes it is unclear whether, like mice, human bone marrow microvasculature is also affected. Spinetti et al show that bone specimens from diabetes patients do indeed exhibit decreased capillary densities and also contain fewer progenitor cells. The team found that while impaired blood supply did contribute to the reduction in progenitor cell populations, high glucose itself also caused these cells to upregulate a pro-apoptosis pathway. This involved inactivation of microRNA miR-155, and the upregulation of its target - the transcription factor, FOXO3a. Taken together, the data suggest that specific treatments designed to preserve the integrity of capillaries of the bone marrow and the function of progenitor cells could prove important for managing diabetes.

Progressive degeneration and weakening of the aortic walls can lead to dilation (aneurysm), tearing (dissection), and even rupturing of the aorta. Although aortic aneurysms are associated with a loss of smooth muscle cells and destruction of the extracellular matrix in the artery wall, little else is known about the pathology of this potentially deadly condition. The protein kinase AKT2 is known to promote the proliferation and migration of smooth muscle cells in vessel walls. While this activity can be harmful in conditions such as atherosclerosis—where it may cause vessel narrowing—Shen and colleagues wondered if it could protect against the weakening of the aortic wall. They found that mice lacking AKT2 had thinner aortic walls and when treated with angiotensin II were more prone to aortic aneurysm, dissection, and rupture. Furthermore, aortic tissues taken from patients with aneurysms and dissections exhibited lower levels of AKT2 than control tissues. Rather than promoting smooth muscle cell proliferation, as initially thought, however, AKT2 appears to protect aortic walls by not only inhibiting production of MMP-9, an enzyme that breaks down extracellular matrices, but also by promoting the production of TIMP-1, an inhibitor of MMP-9. Thus both AKT2 and these downstream factors could be targets for the development of novel aneurysm therapies.

While dihydropyridine calcium channel blockers, such as nifedipine and nicardipine, are often prescribed for pulmonary arterial hypertension (PAH), they are effective in only 15–20% of patients, who are referred to as vasoreactive responders. These drugs work by blocking calcium channels in vascular smooth muscle cells, which lowers cytosolic calcium levels and prevents contraction. Yamamura et al have discovered that these drugs also activate a second type of calcium protein in smooth muscle cells called the calcium sensing receptor (CaSR). And they show that activation of this receptor actually increases cytosolic calcium. In normal vascular smooth muscle cells the expression level of CaSR proteins is too low for this counteraction to be problematic, which explains why such drugs are successful vasodilators for many other conditions. In PAH patient smooth muscle cells, however, CaSR proteins tend to be upregulated. The findings not only offer an explanation as to why so few PAH patients respond to dihydropyridine channel blockers, but they also suggest that by raising smooth muscle cell calcium levels, these drugs could actually worsen PAH. Alternative options, such as non-dihydropyridine calcium channel blockers and CaSR blockers could prove to be more effective for the treatment of PAH, say the authors.

Titin is a large elastic protein found in cardiomyocytes that controls the stiffness of these cells, and as a result the myocardium as a whole. The protein is phosphorylated by many different kinases, and Hamdani and colleagues now add the Ca2+/calmodulin-dependent protein kinase-II (CaMKII) to that list. By performing mass spectrometry of heart tissue from wild type mice and CaMKII-null and overexpressing mice, the team identified several different CaMKII phosphorylation sites in the spring elements of titin. They also showed that phosphorylation of titin by CaMKII reduced the passive force, or stiffness, of cardiomyocytes, which would be expected to facilitate heart filling during diastole. But the authors warn that excessive phosphorylation of these sites might be harmful. Indeed, they found that heart tissue from patients with end-stage heart failure exhibited significantly increased phosphorylation at 4 of the 5 CaMKII sites. CaMKII expression and activity were also increased in failing human hearts, consistent with previous reports linking CaMKII upregulation with heart failure. The authors therefore suggest that manipulating phosphorylation of titin at CaMKII sites could help alter myocardial stiffness and improve overall cardiac function in heart failure patients.

Macrophage abundance in atherosclerotic plaques is strongly associated with the risk of plaque rupture, which can cause thrombus formation and even myocardial infarction or stroke. Therefore, it is important to image and quantify macrophages in human plaques to identify high-risk patients and to inform therapeutic interventions. However, existing imaging techniques lack sufficient sensitivity. Therefore, Majmudar and colleagues set out to develop a viable alternative. They generated radioactive dextran nanoparticles that could be efficiently taken up by macrophages. They injected these nanoparticles into the tail veins of mice and, after allowing time for phagocytosis, they tracked their vascular distribution using a combination of positron emission tomography (PET)—to detect the radioactive signal—and MRI—to determine anatomical location. They found that compared with wild-type mice, mice that were prone to atherosclerosis exhibited considerably higher nanoparticle PET signals from their aortas. The team also found that preventing the recruitment of macrophages to inflammation sites lowered these aortic PET signals. Because nanoparticles used in the study were biodegradable, this technique could perhaps be used to monitor atherosclerotic plaques in humans, say the authors.

Wantha et al show how neutrophils recruit monocytes to sites of inflammation.

It is currently believed that during early stages of acute inflammation, soluble granule proteins released from neutrophils are required for subsequent recruitment of monocytes to the inflammation site. However, the underlying mechanisms remain unclear. Wantha and colleagues investigated the monocyte-attracting ability of cathelicidin and cathepsin - two neutrophil granule proteins conserved between mice and humans. They found that deletion of cathelicidin in mice reduced the recruitment of classical monocytes to inflammation sites. Mice lacking cathepsin, on the other hand, showed no such defect. In vitro experiments using cathelicidin treated human vascular endothelial cells confirmed that the protein is indeed able to recruit classical monocytes. By blocking different receptor proteins on the surface of monocytes, the team discovered that one specific receptor was responsible for mediating cathelicidin-dependent recruitment. Interaction between cathelicidin and this receptor, FRP2, induced the activation of integrins that enable monocytes to adhere to sites of inflammation. This interaction could thus be targeted to treat for a variety of inflammatory disorders, say the authors.

Penn et al report the outcome of a Phase I gene therapy trial for the treatment of ischemic heart failure.

The tissue damage that occurs after a myocardial infarction can lead to progressive heart failure and, if left untreated, to death. Mending damaged cardiac tissue with the help of stem cells is the goal of several current therapeutic investigations. One such approach, adopted by Penn and colleagues, is to recruit the body’s own stem cells to the heart. Stromal cell-derived factor 1 (SDF-1) is a naturally occurring cytokine that attracts stem cells and is upregulated in injured tissues. In the infarcted heart tissue, however, SDF-1 action lasts less than a week, after which stem cell recruitment diminishes. Artificially maintaining SDF-1 expression in damaged myocardium has been shown to improve cardiac function in a number of pre-clinical studies. Penn et al now provide preliminary evidence that the same approach works in patients too. They injected a non-viral DNA vector encoding the SDF-1 gene into the peri-infarcted myocardia of 17 heart failure patients. Four months later, these patients exhibited improvements in quality of life, 6 minute walking distance, and in the overall clinical classification of their condition. And 12 months later, these improvements persisted. Although this was a relatively small Phase I trial, the results indicate that a larger, randomized trial is warranted.

As new blood vessels form, endothelial cells must not only migrate into their appropriate positions, but also arrange themselves so that their apical surfaces face the vessel lumen. In other cell types, migration and orientation are controlled by factors called polarity proteins, but surprisingly few studies have examined the role of these factors in endothelial cells. Michaelis and colleagues have now discovered that silencing the polarity protein, Scrib, in cultured vascular endothelial cells disrupts cell orientation, directed migration, and vessel formation. Their immunoprecipitation studies reveal that Scrib interacts with integrin α5, a transmembrane protein responsible for binding the extracellular matrix and regulating cell migration. Scrib not only promoted the cell surface expression of integrin 5, but also prevented its lysosomal degradation. In addition, the team showed that either the knock-out of Scrib in mouse embryos or the knock-down of Scrib in zebrafish embryos caused both the delay and disruption of blood vessel development. This newly identified role in angiogenesis could make Scrib a promising target for both pro- and anti-angiogenic treatments, say the authors.

Contrary to popular belief, creatine may not be essential for high-energy performance, say Lygate et al.

Creatine, a molecule abundant in heart and skeletal muscle cells, is dramatically reduced in the failing heart. And because creatine converts readily to and from phosphocreatine, it is thought to be a major transporter and source of phosphate for ATP production. In addition, mice lacking creatine have stunted maximal heart muscle activity and recover poorly if at all from myocardial infarction. Such observations have led to the conclusion that even though creatine may not be necessary for basal muscle activity, it is necessary for high-energy expenditure, particularly under conditions of stress. However, Lygate and colleagues now challenge that view. They say the method for eliminating creatine in previous studies might have caused off-target effects. To address this issue, the team engineered mice that lacked an essential creatine production enzyme. Surprisingly, these mice, although 100 percent creatine-free, could run just as far and just as fast as wild type mice. They also exhibited the same capacity for exercise on a treadmill and showed equivalent recovery and survival after myocardial infarction. These findings raise the question, if creatine is not required for high energy performance, why is it present in high-energy cells? The authors suggest creatine may play, an as yet undetermined role, in longer-term energy production.

Milroy disease is an inherited condition in which congenital abnormalities in the lymphatic vessels prevent the proper drainage of tissue fluid, causing characteristic swelling in the legs and feet. Although the most common cause of Milroy disease is a mutation in the vascular endothelial growth factor receptor 3 (VEGFR3), nearly 30 percent of patients do not carry such a mutation. To search for alternative causes, Gordon and colleagues performed whole exome sequencing on five Milroy disease patients that were free of mutations in VEGFR3. One of these patients showed a mutation in a VEGFC, a ligand of VEGFR3. This mutation caused a translational frameshift that generated a truncated protein. Three members of this patient’s family also displayed Milroy-like lymphedema, while carrying the very same VEGFC mutation. Functional studies in zebrafish showed that while wild type VEGFC protein induced the sprouting of both blood and lymph vessels, the mutant truncated form of the protein did not. In light of these results, the authors suggest that patients who present with Milroy disease symptoms without a VEGFR3 mutation should be screened for mutations in VEGFC.

Atherosclerosis is a leading cause of death in patients with type 2 diabetes. But exactly how high blood sugar and insulin resistance are linked to the development of atherosclerosis is unclear. It is known that insulin signaling in myeloid cells, such as macrophages, is mediated by a family of transcription factors called FoxO. Given that macrophages are a principle cell type in atherosclerotic plaques, Tsuchiya and colleagues removed FoxO factors from these cells and examined the effect on atherogenesis. They found that atherosclerosis-prone mice lacking FoxO factors in their myeloid cells had larger plaques that contained increased numbers of macrophages. The cause of this amplified lesion formation appeared to be bipartite: First, increased proliferation and decreased apoptosis contributed to the overabundance of myeloid cells in the lesion. And second reactive oxygen species, which are known to accelerate atherogenesis, were more abundant in the FoxO-lacking cells. Importantly, Tsuchiya et al showed that treating these mice with an antioxidant decreased myeloid cell numbers and lesion formation. Further studies on FoxO could aid in the design of effective anti-atherosclerosis treatments for diabetic patients.

Unlike cardiac and skeletal muscle cells, vascular smooth muscle cells (VSMCs) are not terminally differentiated. Instead, they retain the ability to proliferate and migrate when necessary, such as when blood vessels require regrowth after an injury. However, VSMC proliferation and migration can sometimes lead to an overgrowth of the cells, which may contribute to neointima formation, atherosclerosis or other vascular problems. This switch from a resting to a proliferative and migratory phenotype has been associated with the upregulation of calcium channel component Orai1, and inhibiting Orai1 upregulation has been shown to prevent neointima formation in mice. The bad news, however, is that Orai1 functions in multiple cell types, making it a non-specific target for potential therapies. Gonzales-Cobos and colleagues thus investigated Orai 3, a lesser-known homologue of Orai1. They found that, like Orai1, Orai3 was upregulated in proliferative and migratory VSMCs, but, it created an alternative form of calcium channel. Even though the channels differed, the effect of suppressing Orai3 expression in mice was the same, that is, neointima formation was prevented. Thus, for neointima-directed therapies, Orai3 may prove to be a better target than the ubiquitous Orai1.

As we age our arteries tend to stiffen, which can accelerate the process of cardiovascular degeneration. This vessel stiffening has been largely attributed to changes in the extracellular matrix of the arterial wall. But recent observations suggest that vascular smooth muscle cells (VSMC) surrounding the blood vessels also stiffen with age. To study the effect of changes in VSMC stiffness in living mice Galmiche and colleagues increased and decreased the expression of serum response factor (SRF) in these cells. SRF is a transcription factor that is expressed in many cell types. In VSMCs, however, it controls the expression of cytoskeletal and contractile proteins. The team found that mice lacking SRF in VSMCs exhibited lower blood pressure and higher elasticity of the carotid artery than wild type mice. While this increase in elasticity was related to the expected decrease in the expression of contractile and cytoskeletal proteins, it was also linked to a weaker interaction between the VSMCs and the extracellular matrix. Mice over-expressing SRF in their VSMCs, on the other hand, showed increased VSMC contractility. These findings suggest that VSMCs could be an important target for preventing age-related arterial stiffness and the problems that go along with it.

The renin-angiotensin system (RAS) controls the body’s fluid levels and blood pressure. Many components of the system are known and novel participants in the network are being discovered. Lautner et al studied one such novel RAS hormone called alamandine and discovered that this heptapeptide is almost identical to another RAS component—the vasodilator angiotensin-(1–7). The 2 proteins differ by just one amino acid. Indeed, alamandine could be synthesized from angiotensin-(1–7) by decarboxylation of the amino-terminus aspartate into alanine—though, as the team showed, alamandine could also be produced by catalytic hydrolysis of angiotensin A. Like angiotensin-(1–7), alamandine induced vasodilation by promoting the release of nitric oxide from cells, but despite the close sequence and functional similarity of the two proteins, they acted through different receptors. Angiotensin-(1–7) binds a receptor called Mas, where alamandine was found to bind to MrgD—previously identified as a poor angiotensin-(1–7) receptor. Importantly, treatment of hypertensive rats with alamandine resulted in a long-lasting reduction in blood pressure. These findings not only lend insight into the complexities of the renin-angiotensin system, but may also drive the development of future anti-hypertensive therapies.

Bhargava et al devise a new trick to analyze the location and function of ion channels in living cells.

Patch-clamp recording of a cell measures the activity of ion channels, but reveals nothing as to the channels’ whereabouts. Scanning ion conductance microscopy (SICM), on the other hand, provides a detailed cell surface topography, but no information about channel activity. Given that both methods work by detecting the flow of ions into a pipette—providing a direct readout of channel activity in the case of patch-clamp, and acting as an indicator of the distance from the cell surface in SICM—researchers had hoped to combine the two and thus measure channel activity at defined locations. But there was a problem: SICM requires a pipette tip no more than 100nm in diameter, while patch-clamp pipettes should be approximately 300nm. Bhargava’s new ingenious approach is to first perform SICM with a fine-tipped pipette, then move the pipette away from the sample and tap it repeatedly against the bottom of the dish until it snaps to a diameter appropriate for patch clamp analyses—the tapping and snapping being tightly controlled by a piezo device. The snapped, or clipped, pipette is then returned to the previously scanned site for patch clamp recordings. Et voila, by one simple maneuver, researchers can go from spatial to functional analysis of their channel of interest.

An anti-malarial drug could be effective against pulmonary arterial hypertension, report Long et al.

Pulmonary arterial hypertension (PAH) leads to an increase in pulmonary arterial smooth muscle cells (PASMCs) proliferation, while decreasing apoptosis. This causes the walls of the arteries to thicken and the blood pressure to increase. The increase in PASMC proliferation has been linked to a loss-of-function of an inhibitor of proliferation called BMPR-II. Indeed, approximately 70% of heritable cases and 10–40% of sporadic cases of the PAH are caused by mutation in this gene. Even in cases of PAH without BMPR-II mutation, expression of the protein may be reduced. BMPR-II is expressed on the surface of cells and gets degraded via the lysosomal system. Therefore, Long et al therefore considered whether preventing this turnover might help maintain BMPR-II activity and slow down the progression of PAH. They treated PASMCs in culture with the anti-malarial drug chloroquine, which is a known inhibitor of lysosomal activity and has the added benefit of inducing apoptosis. Sure enough, they found that there was a decrease in PASMC proliferation, and apoptosis increased. They also found that in a rat model of PAH, injection of chloroquine prevented pulmonary arterial wall thickening and curbed PAH progression. These promising results suggest that chloroquine could be tested clinically for the treatment of PAH patients.

The Ser-Pro and Thr-Pro motifs in proteins are recognized and phosphorylated by proline-directed kinases such as Akt and MEK. These phosphorylation events are like light switches that can turn the target proteins on or off. But, the lights can also be dimmed or brightened by Pin1, a small protein that binds to phosphorylated Ser-Pro and Thr-Pro and adjusts the intensity of activation or repression. Pin-1 thus fine tunes a variety of cellular signaling pathways. However, little is known of the protein's activity in cardiomyocytes. Toko and colleagues discovered that Pin1 is highly expressed in the newborn mouse heart, but that its expression decreases by almost half as mice age. An exception to this phenomenon was pressure overload, when the expression of Pin1 was increased dramatically. Investigating, how different levels of Pin1 affected pressure-induced hypertrophy, the team found that mice lacking Pin1 maintained better cardiac function, exhibited fewer signs of hypertrophy and were more likely to survive following pressure overload than their wild type counterparts. Surprisingly over-expression of Pin1 also attenuated hypertrophy. The team deciphered that both under and over-expression of Pin1, via different pathways, reduced the activity of MEK—a known instigator of hypertrophy. Tweaking Pin1 balance in either direction might be beneficial in treating hypertrophy, say the authors.

Accumulating evidence suggests that the heart does not solely consist of terminally differentiated cells, but that it could also undergo regeneration. Indeed, a number of cardiogenic cell types have been discovered in the heart and in the bone marrow. The existence of these cells has sparked excitement regarding their potential use in reparative therapies. But whether these cardiogenic cells are in anyway related to one another has not been established. Dey et al isolated three populations of mouse progenitor cells from the heart—ckit+, Sca1+ and SP—and two from the bone marrow—ckit+ and MSCs. They extracted RNA from each and compared their transcriptional profiles. They found that cardiac-derived progenitors and bone marrow-derived progenitors were very different from each other, with the cardiac-derived cells tending to express higher levels of matrix and cell-to-cell attachment factors, and the bone marrow cells expressing higher levels of cell cycle factors. Even the two ckit+ populations differed substantially. Of the three cardiac populations, the team determined that Sca1+ cells were the most closely related to differentiated cardiomyocytes, while the ckit+ cells were the most primitive, suggesting the three progenitor types might represent distinct differentiation stages. The improved understanding progenitor population relatedness should help in designing future cardiac repair strategies, say the authors.

Zhao et al figure out how adiponectin improves the action of insulin in muscle.

The hormone adiponectin circulates in the blood and is known to enhance insulin action. Indeed, low plasma levels of adiponection are associated with insulin resistance and diabetes in humans and mice, while replenishing adiponectin levels in mice improves insulin sensitivity. Adi ponectin also promotes nitric oxide production in endothelial cells, and thus induces vasodilation. Now Zhao and colleagues show that it is this vasodilatory activity that enables adiponectin to improve insulin sensitivity. They found that injecting rats with adiponectin increases plasma nitric oxide levels, consequently increasing blood volume in the hindlimb skeletal muscle. Insulin uptake into the muscle also increased as did the rate of whole-body glucose disposal in response to insulin administration. These effects of adiponectin were blocked by inhibiting nitric oxide synthase—the enzyme responsible for producing nitric oxide—confirming that vasodilation was required for the increased insulin sensitivity. The authors explained that vasodilation expands the endothelial exchange surface for uptake of insulin, and suggest that targeting the muscle vasculature and hypoadiponectinemia could be important for the prevention and treatment of insulin-resistance and type 2 diabetes.

Sudden cardiac death caused by ventricular fibrillation claims the lives of approximately 300,000 North Americans each year, and in nearly 10% of cases the cause is unknown, or idiopathic. Recently, members of a Dutch family with idiopathic ventricular fibrillation (IVF) were found to over-express transcripts of the DPP6 gene. Because the DPP6 protein modulates potassium channel kinetics and trafficking, Xiao and colleagues questioned whether high levels of DPP6 might actually affect potassium channel function and, in turn, cause arrhythmia. The team found that over-expression of DPP6 in canine heart tissue did indeed affect channel function, causing an increase in the density of transient outward potassium currents. Interestingly, this increase occurred in Purkinje cells, and ventricular myocytes were unaffected. While DPP6 was responsible for binding potassium channels in Purkinje cells, in ventricular cells a protein called KChIP2 performed this function, explaining the differing results. Computer modeling suggested that the increased current density in Purkinje cells would shorten their action potentials and initiate early repolarization as well, which would indirectly promp arrhythmia in adjacent ventricular myocytes. Altogether these findings have the potential to improve not only the understanding of ventricular arrhythmia mechanics, but possible arrhythmia treatments, as well.

The increased sensitivity of cardiomyocytes to calcium has long been associated with hypertrophic cardiomyopathy, as well as other forms of heart disease. This extra sensitivity increases the risk of arrhythmia, and subsequently of sudden cardiac death. It is not clear how calcium sensitivity and arrhythmia are linked, but it is known that gap junctions—connections between neighboring cells—are crucial for the propagation of electrical currents across the myocardium. So Huke and colleagues examined gap junctions in myofilaments that were hypersensitive to calcium. They found that when these myofilaments were stressed, the gap junction protein Cx43 rapidly de-phosphorylated, causing conduction velocity to slow and neighboring cells to lose their electrical coupling—which would thus result in arrhythmia. Dephosphorylation of Cx43 was accompanied, and most likely caused, by depleted ATP levels in the immediate gap junction vicinity. This ATP depletion was perhaps due to the high energy requirements of calcium sensitive cells, the authors suggest. Whatever the reason, reversing calcium sensitivity prevented both localized ATP depletion and aberrant electrical conduction. This newly discovered proarrhythmic mechanism may help guide the development of antiarrhythmic therapies, say the authors.

Obesity is often associated with inflammation of the adipose tissues, which exacerbates insulin resistance and increases the risk of cardiovascular disease. The adipocytes are thought to recruit monocytes and macrophages via the generation of reactive oxygen species (ROS), which in turn promote the production of immune cell chemoattractants, such as monocyte chemotactic protein-1 (MCP-1) and serum amyloid A3 (SAA3). Both apoliporotein A-1 (apoA-1) and high density lipoprotein (HDL) are known to reduce inflammation in endothelial cells and macrophages, but their role in modulating inflammation in adipocyte tissue was unknown. Umemoto and colleagues show that in cultured adipocytes, ApoA-1 and HDL inhibit the production of reactive oxygen species which caused a reduction in gene expression of Mcp-1 and Saa3. Furthermore overexpression of human apoA-1 in mice fed a high-fat diet also reduced the recruitment of immune cells to adipose tissue. The new information provided in this paper should be helpful in guiding the design of therapies that prevent inflammation in adipose tissue, and thus the cardiovascular complications that go with it.

Smooth muscle cells (SMCs) are an essential component of blood vessels, and are thus required to engineer functional blood vessels in culture. Tissue engineers need a reliable and plentiful supply of SMCs. However, the methods currently used to obtain these cells - such as retrieving them from adult arteries and expanding them in vitro, or differentiating them from embryonic stem cells - can be inefficient, arduous, or both. Karamariti and colleagues propose a new approach, which involves direct reprogramming of fibroblasts into SMCs, by transfecting human fibroblasts with factors used for making induced pluripotent stem (iPS) cells. But instead of fully dedifferentiating the cells, they plated partially dedifferentiated cells, called PiPS, on collagen IV and induced them to become SMCs. The resulting cells exhibited SMC morphology, expressed SMC-specific markers and were capable of contracting like native SMCs. Through a careful monitoring of the reprogramming process, the researchers also discovered a novel factor, called DKK3, which activated the expression of an important SMC marker. The reprogrammed cells were then used with endothelial cells to build tissue-engineered vessels that functioned like their native counterparts when transplanted into mice. Overall, this new method could improve current approaches to vessel engineering and, as a result, help in the development of vessel grafts for patients.

High HDL levels in blood are associated with a lower risk of heart disease. The protective effects of HDL have been linked, in part, to its ability to promote cholesterol efflux from macrophages. However, it is unclear whether this efflux is indeed protective. Cholesterol efflux from macrophages is controlled by two transporter proteins, ABCA1 and ABCG1, so Westerterp and colleagues specifically deleted these proteins from the macrophages of atherosclerosis-prone mice. They found that this procedure led to an increase in the lipid content of the macrophages and increased atherogenesis. And while a deficiency in the cell’s cholesterol efflux did play a role in these changes, it was not the only contributing factor. In fact, in mice lacking ABCA1- and ABCG1 transporters in macrophages there was also an increase in monocytes and neutrophils, which led to increased inflammation in atherosclerotic plaques. It turned out that the ABCA1- and ABCG1-deficient macrophages were producing high levels of the cytokines G-CSF and M-CSF, which promote the production of neutrophils and monocytes, respectively. These findings suggest that strategies aimed at promoting cholesterol efflux will prevent the pro-inflammatory and pro-atherogenic potential of macrophages, and furthermore, that monitoring G-CSF and M-CSF levels could help assess the success of such strategies.

Inhibition of ApoC-III by antisense RNA reduces triglyceride levels in both animals and humans.

Elevated levels of triglycerides in the blood are a well-known risk factor for coronary heart disease. And, therefore, reducing triglyceride levels might reduce that risk. To lower dangerously high triglyceride levels, doctors currently prescribe statins, niacin, fibrates and omega-3-fatty acids, but in many patients, achieving the recommended level is difficult. More effective remedies are therefore required. The glycoprotein ApoC-III is an important regulator of plasma triglyceride, and high levels of ApoC-III are associated with high levels of triglycerides. High ApoC-III levels have also been independently associated with metabolic disorder and type 2 diabetes. Thus, Graham and colleagues investigated lowering ApoC-III levels with antisense nucleotides directed against human and rodent ApoC-III mRNA. The antisense nucleotides successfully reduced plasma levels of both ApoC-III and triglycerides in rats, mice, transgenic mice carrying the human ApoC-III gene, rhesus monkeys, and most importantly, in healthy humans. Although this phase-I trial was performed in just three subjects, the results were significant and there were no adverse safety indications. The findings suggest that larger-scale phase-II clinical trials are warranted for this first-of-its-kind antisense ApoC-III therapy.

Ischemia-reperfusion, which occurs in a number of clinical settings—such as during coronary angioplasty or organ transplant—can induce inflammation causing damage to the reperfused tissue. This inflammation is in part controlled by the activation of the transcription factor NF-κB, which in turn activates inflammatory genes. The protein Cezanne is a deubiquitinating enzyme and a known negative regulator NF-κB. Luong et al now report that a substrate of Cezanne is a protein called TRAF6, the polyubiquitination of which activates NF-κB and the inflammatory signaling. The team found that the silencing of Cezanne in cultured cells led to greater TRAF6 ubiquitination, while the deletion of Cezanne in mice increased injury and inflammation following ischemia-reperfusion. Interestingly, the team also found that the mRNA and protein levels of Cezanne were increased following hypoxia and reoxygenation in both cultured cells and animals, suggesting that Cezanne is automatically activated to dampen the effects of NF-κB and prevent over-inflammation. Artificially boosting Cezanne activity could further reduce inflammation and thus serve as an effective clinical strategy to prevent reperfusion injury, suggest the authors.

Ramirez et al identify a microRNA that reduces cholesterol export from macrophages as well as the body.

The transporter protein ABCA1 is essential for removing cholesterol from macrophages and it helps in preventing these cells from developing into foam cells that form atherosclerotic lesions. In the liver, ABCA1 controls the biogenesis of HDL, which promotes cholesterol removal from the blood for excretion in bile. A number of microRNAs (miRs) have been shown to regulate ABCA1, and now Ramirez and colleagues have identified miR-144 as a new ABCA1 regulator through an unbiased screen for miRs upregulated in macrophages that had been stimulated to undergo cholesterol efflux. They found that ABCA1 mRNA has seven potential binding sites for miR-144, and that over-expressing miR-144 in macrophages and in mice suppressed ABCA1 expression, confirming that miR-144 and ABCA1 mRNA do indeed interact. Importantly, the inhibition of miR-144 by an antisense oligonucletide not only increased ABCA1 levels and the efflux of cholesterol in macrophages, but also increased HDL levels in mice, thereby potentially boosting cholesterol export in two ways. Thus, miR-144 inhibition could be a novel therapeutic strategy for lowering cholesterol.

de Aguiar Vallim et al independently identify microRNA-144 as a regulator of ABCA1.

At the same time when Ramirez and colleagues were screening macrophages for microRNAs (miRs) related to cholesterol efflux, de Aguiar Vallim and his team were searching for miRs that might be involved in lipid and lipoprotein metabolism in the liver. And both teams independently identified miR-144. Initially, de Aguiar Vallim’s team identified two miRs—miR-144 and miR-451. Both were highly conserved and also co-transcribed in response to hepatocyte stimulation with FXR—a transcription factor that regulates lipoprotein metabolism, among other things. But in a search for putative targets of miRs-, only miR-144 yielded strong, highly conserved candidates. And at the top of the list was ABCA1. The team over-expressed miR-144 in human and mouse hepatocytes and found that, ABCA1 protein levels were suppressed in both these cells. Similarly, the over-expression of miR-144 in mice decreased ABCA1 in the liver and HDL in the blood. Silencing of miR-144 with antisense oligonucleotides, on the other hand, increased ABCA1 and HDL levels, just as Ramirez and colleagues observed. Although FXR induced miR-144, the transcription factor has also been reported to reduce atherosclerosis in mice, suggesting FXR's particular role in cholesterol efflux is complex and requires additional study.

Circulation Research, vol 113, 2013

Kang et al uncover the role of apelin-APJ signaling in mammalian cardiovascular development.

The small peptide ligand apelin and its receptor APJ are known to be involved in cardiovascular development in both frogs and fish. Surprisingly, the role of apelin-APJ in mammalian cardiovascular development remains unknown. Kang et al studied mice that had homozygous null alleles of the Apj gene, most of whom die as embryos between stage E10.5 and E12.5 of embryogenesis. They found that the mutant mouse embryos exhibited a broad spectrum of cardiovascular developmental abnormalities, including particular blood vessels that were either poorly developed or entirely missing, and hearts that were malformed. Even the few mice—less than 10 percent—that survived into adulthood showed significant heart malformations, including enlarged right ventricles and septal defects. Apelin-APJ regulates the expression of the transcription factor KLF2, and the team showed that this occurs via activation of another transcription factor - MEF2, a known activator of KLF2. The critical importance of apelin-APJ signaling in mammalian cardiovascular development suggests that the ligand and its receptor could be a focus for study in humans with congenital heart abnormalities, the authors say.

Vascular smooth muscle cells (VSMCs) are generally quiescent in adult blood vessels but can be sparked back into a proliferative state both in atherosclerotic lesions and during vessel injury, where they promote angiogenesis leading to an increase in angiogenesis. The microRNA miR-126 is a regulator of angiogenesis and it is produced by endothelial cells (ECs). Given the proximity of ECs to SMCs in vessel walls, Zhou and colleagues wondered whether miR-126 might promote angiogenic behavior in SMCs. And It did. They found that SMCs cultured with ECs exhibited higher levels of proliferation, and this increase was mitigated when miR-126 was blocked in the ECs. Interestingly, ECs exposed to laminar sheer stress did not secrete as much miR-126 as those exposed to an oscillatory fluid flow, which is known to promote atherogenesis in blood vessels. Indeed, endothelial material extracted from mouse arterial regions associated with oscillatory flow and atherosclerotic lesions had a 3-fold higher level of miR-126 expression than arterial regions experiencing lamina flow. These results provide at least a partial explanation of why oscillatory flow promotes atherosclerosis, and may offer clues to stop SMCs from undergoing pathological proliferation.

Clearing up the remains of apoptotic cells is essential for wound healing and for the resolution of inflammation. Indeed, dysfunctional cell clearance is commonly observed in chronic inflammatory disorders. Macrophages do most of this work, using receptors on their cell surface to recognize, and trigger phagocytosis of apoptotic cells. However, the receptors can be proteolytically cleaved from macrophages, and this has been offered as one reason why levels of soluble receptors are increased in the tissue fluids during inflammation. To investigate this phenomenon more closely, Driscoll and colleagues deleted ADAM17 - a protease known to cleave cell surface receptors involved in inflammation—from macrophages in mice. Following induced inflammation, the team observed that phagocytosis of apoptotic cells was increased in mice with ADAM17-null macrophages, and that inflammation resolved more quickly. They also identified CD36 as the cell surface receptor targeted by ADAM17, showing that soluble CD36 levels were reduced in media incubated with ADAM17-null macrophages compared with wildtype macrophages. This work provides a new explanation of why soluble CD36 levels are increased in many chronic inflammatory conditions, and these findings may also provide additional avenues for resolving inflammation.

Coronary angioplasty, whereby doctors mechanically widen narrowed blood vessels can restore blood flow to the heart and improve symptoms of coronary heart disease. However, the procedure can also injure the vessel wall, which promotes the migration and proliferation of vascular smooth muscle cells (VSMCs) and restenosis. Such injury, induced by angioplasty causes the release of inflammatory signals such as prostaglandins, and it has been shown that blocking the activity of prostaglandin E2 (PGE2) or the enzyme COX2, which synthesizes PGE2, attenuates VSMC hyperplasia after vascular injury. Zhang and colleagues now confirm this finding, showing that such VSMC growth is reduced in COX2-knockout mice. But simply blocking COX2 activity in angioplasty patients is not a viable option for therapy, say the authors, because the enzyme also synthesizes prostacyclin, which has cardioprotective effects. The good news is the team also identified the receptor EP3 on VSMCs as the target for PGE2. EP3 antagonists prevented VSMCs from migrating in response to PGE2, suggesting that targeting EP3 might be a good alternative for preventing angioplasty-induced restenosis in patients.

When the body experiences acute hypoxia, arteries and arterioles of the lungs constrict, while in chronic hypoxia these vessels undergo remodeling that involves overt proliferation of endothelial and smooth muscle cells and this can cause chronic pulmonary hypertension. Within the vessel wall cells themselves, hypoxia directly inhibits glucose oxidation, but glucose oxidation can also be indirectly inhibited by stress signals from the endoplasmic reticulum (ER), limiting the transfer of calcium to mitochondria, causing the dysfunction of several respiratory enzymes. The protein UPC2 transfers calcium from the ER to mitochondria, and now Dromparis and colleagues have found that UPC2 deficiency leads to pulmonary artery remodeling and pulmonary hypertension in mice, even in the absence of hypoxia. Within the pulmonary artery smooth muscle cells, loss of UPC2 caused reduced mitochondrial calcium levels, respiratory enzyme dysfunction, and induction of the transcription factor hypoxia inducible factor 1α. Thus at both the tissue and cell level, UPC2 deficiency closely mimics hypoxia. For this reason, UPC2-lacking mice could serve as a useful model for future investigations into the pathological pathways that cause pulmonary hypertension in humans, say the authors.

Inhibiting CETP activity may help diabetes patients as well as those with atherosclerosis, say Siebel et al.

Cholesterylester transfer protein (CETP) is a plasma protein that decreases plasma HDL levels. Therefore CETP inhibition may be one approach for increasing HDL levels in the plasma. Indeed, several on-going clinical trials are investigating the use of CETP inhibition to raise HDL levels and attenuate atherosclerosis. Although the results to date have not been encouraging, circumstantial evidence from these trials with this class of drugs indicates that they might be beneficial by improving glucose homeostasis in patients with type 2 diabetes. To examine the potential anti-diabetic effects of CETP inhibition, Siebel and colleagues have performed a small placebo-controlled trial in 25 healthy males to investigate whether CETP inhibition could increase insulin secretion. They found that after 14 daily doses of a CETP inhibitor, post-breakfast insulin levels in the subjects were raised. Furthermore, mouse pancreatic β cells incubated with plasma from the study subjects who received the CETP inhibitor secreted greater amounts of insulin than those incubated with plasma from the placebo-control subjects. These findings suggest that independent of their potential anti-atherosclerotic effect, CETP inhibitors could be an effective treatment for type 2 diabetes, but larger trials are needed to explore this potential therapeutic use.

Angiotensin II (Ang II) is a hormone that promotes vasoconstriction and hypertension, as well as inflammation, fibrosis and the growth of vascular smooth muscle cells (VSMCs). A number of genes and microRNAs (miRs) have been identified in VSMCs that respond to Ang II treatment, controlling proliferative, inflammatory, and fibrosis pathways. But, recent findings have indicated that longer non-coding RNAs can also regulate cell responses. This led Leung and colleagues to investigate whether other forms of RNA are regulated by Ang II. Comparing the transcriptomes of Ang II–treated and non-treated rat VSMCs, they discovered 491 differently expressed transcripts including 24 novel long non-coding RNAs and 14 novel protein-coding transcripts. Upon further examination, the team found many of the non-coding RNAs were located in close proximity to other Ang II–regulated transcripts. The expression of one particular RNA called Lnc-Ang362 closely resembled that of two miRs previously implicated in Ang II–induced VSMC proliferation. In fact, Lnc-Ang362 actually contained the two miRs. Furthermore, suppressing Lnc-Ang362 also suppressed VSMC proliferation. Lnc-Ang362 and the other novel transcripts could be targets for therapy in hypertension and other Ang II–related disorders, the authors suggest.

Stem cell therapy remains a promising technique for improving cardiac function after myocardial infarction. Thus far, however, the majority of experimental stem cell transfers have produced only marginal recovery. Part of the problem is that the engrafted cells do not survive in their new environment for very long. The protein PHD2 is a negative regulator of two different survival factors—HIF-1α and NF-κB—leading Wang et al to investigate whether suppressing PHD2 activity in stem cells might improve their survival in the heart. The team used RNA interference to inhibit PHD2 in human adipose-derived stem cells and compared the heart-mending capability of these cells with non-treated cells in mice that had suffered myocardial infarctions. Four weeks after transferring the cells, mice that had received PHD2-silenced stem cells showed better left ventricle function, including improved ejection fraction, less fibrosis, and a smaller infarcted area. In vitro studies revealed that the PHD2-silenced cells secreted increased levels of an anti-apoptotic factor and the growth factor IGF-1, which might explain the improved performance of these cells. Thus, PHD2 silencing could prove a successful strategy for enhancing the efficacy of current stem cell therapy.

Because of advances in medical therapy, more patients now survive heart attacks than before, but the number of them that go on to develop life-threatening heart failure is on the rise. Biomarkers for predicting which patients are at risk of heart failure would therefore be extremely beneficial. It was discovered recently that microRNAs (miRs) can be secreted from a variety of tissues and can circulate stably in the blood—protected inside tiny membrane-bound vesicles called exosomes. Matsumoto and colleagues investigated whether the levels of any circulating miRs in patients with myocardial infarction are predictive of future heart failure. They collected blood from patients between two and three weeks after myocardial infarction and documented the expression levels of multiple miRs. One in particular, miR-192, was expressed more highly in those patients that went on to develop heart failure within a year. miR-192 is regulated by the tumor suppressor protein p53, and the team discovered that two additional p53-regulated miRs, miR194 and miR34a, were also highly expressed in the patients that would later develop heart failure. These three miRs might therefore be useful for predicting, monitoring, and treating heart failure.

Cyclic GMP (cGMP) is an important signaling molecule that controls a number of cardiovascular processes. Indeed, dysfunctional cGMP signaling has been linked to arterial hypertension, angina and more. But investigating the role of cGMP in cardiovascular physiology is an arduous task because the molecule is difficult to monitor in live cells and tissues. To circumvent this problem, Thunemann and colleagues developed a mouse that expresses a fluorescent biosensor of cGMP. This sensor consists of a cGMP-binding peptide flanked by two fluorescent proteins—one cyan, one yellow. When not bound to cGMP, excitation of the sensor’s cyan protein leads to a transfer of energy to the yellow protein, making it fluoresce brightly. Binding to cGMP, however, reduces energy transfer, by forcing the two fluorescent proteins further apart, which enhances the cyan and reduces the yellow fluorescence. To test this probe, the team treated smooth muscle cells from the mice with vasodilators to induce relaxation, a process known to be controlled by cGMP. Sure enough, they found the cyan signals increased and yellow signals diminished indicating an increase in cGMP levels. Equivalent experiments looking at whole blood vessels in live mice also showed the vasodilator-induced increase in cGMP; confirming the usefulness of these animals for studying cGMP dynamics in vivo.

Liang et al discover a useful marker for tagging cells of the first heart field.

During embryogenesis, the heart develops from two cell populations called the first and second heart fields. Cells of the first heart field give rise to the early heart tube, and later, to most of the left ventricle as well as parts of the left and right atria. The second heart field by contrast contributes to the right ventricle and outflow tract, and the majority of the two atria. While the transcription factor Is1l is a useful marker of the second heart field, no such marker was identified for the first heart field—until now. The channel protein HCN4 is associated with adult pacemaker cells, but studies of its expression during early development hinted at a first field-like pattern. Liang and colleagues therefore engineered mice to express easily visualized reporters from the HCN4 locus to track HCN4-expressing cells in embryos. They found that HCN4 did indeed mark the first field cells, labeling the early heart tube and left ventricle as expected. They also found that HCN4 is expressed in distinct precursors of the cardiac conduction system and marks the entire system by late fetal stages. Determining the cellular origins of the conduction system will help researchers understand how and why it sometimes fails causing cardiac arrhythmia.

Inhibiting PKCβ2 may reduce the risk of atherosclerosis in patients with diabetes, say Li et al.

Patients with diabetes are at an increased risk for cardiovascular complications, including atherosclerosis. Likewise, diabetic mice prone to atherosclerosis develop more severe atherosclerotic lesions than non-diabetic mice. The molecular details explaining why diabetes worsens atherosclerosis are still being deciphered, but part of the problem seems to be that during diabetes insulin-resistant endothelial cells fail to increase the production of the vasodilator nitric oxide and to suppress production of VCAM-1, which promotes immune cell adherence to vessel walls. Activation of protein kinase C (PKC) β in endothelial cells of patients with diabetes has been shown to inhibit insulin signaling, and now Li and colleagues have investigated the involvement of PKCβ2 more closely. They found that even in the absence of diabetes, overexpression of PKCβ2 in the endothelium of atherosclerosis-prone mice decreased the activation of endothelial nitric oxide synthase in response to insulin and prevented insulin-induced downregulation of VCAM-1. PKCβ2 overexpression also increased production of the vasoconstrictor endothelin-1, thus further antagonizing insulin action. As a result, these mice exhibited larger atherosclerotic lesions in their aortas. Altogether, the results suggest that inhibiting PKCβ2 may be a potential treatment for reducing atherosclerosis in patients with diabetes.

Calcium is the major intracellular regulator of cardiomyocyte contraction. It is released from its intracellular storage site, the sarcoplasmic reticulum, into the cytoplasm where it then activates the contractile machinery. But after each contraction, the cell must relax— a process that requires the prompt and efficient removal of calcium from the cytosol. This combined release and clear up of calcium is known as a calcium transient. It has been shown that the release of calcium across the cell is synchronized to ensure coordinated control of the contractile machinery, but less is known about the clean-up, or decay phase. Hohendanner and colleagues now show that calcium removal occurs at different microdomains throughout the cell and that the speed of decay varies from domain to domain. Furthermore, the rate of relaxation at adjacent contractile machinery varies in concordance with the differing decay rates of the domains. They also found that the degree of dyssynchrony is vastly exaggerated in failing human hearts as well as in mice with hypertrophy and pigs with chronic myocardial ischemia. This, increased dyssynchrony slows down the overall rate of relaxation and, the authors suggest that it might contribute to contractile dysfunction associated with cardiac hypertrophy and heart failure.

While stem cell therapy is a promising approach for repairing damaged hearts, the results obtained to-date have been modest. Part of the problem is that different types of stem cells yield different results and the precise mechanisms underlying the beneficial effects of stem cells remain unclear. Thus, Duran and colleagues examined two mechanisms of repair—differentiation of stem cells into cardiac cell types and secretion of paracrine factors—in two different stem cell types, one from cortical bone and another from the heart. They found that when injected into damaged mouse hearts, the bone cells were actually more adept at differentiating into cardiac cell types—including cardiomyocytes, vascular endothelial cells, and smooth muscle cells—than the heart-derived cells, which only differentiated into immature cardiomyocytes. Cells from the bone also displayed sustained expression of pro-angiogenic paracrine factors, which correlated with better neovascularization of the damaged hearts. Cardiac stem cells, on the other hand, produced similar paracrine factors but only for one day after injection. Importantly, the better performance by the bone cells translated to improved heart function and increased survival for the mice, suggesting that the use of these cells might improve outcomes of cell therapy.

Constriction of vascular smooth muscle cells (VSMCs), which contributes to hypertension, is controlled in part by phosphorylation of the myosin light chain (MLC). This post-translational modification enables actin and myosin molecules of the contractile machinery to interact and initiate contraction. MLC is known to be phosphorylated by MLCK but recent work suggests that at least one other kinase may be involved. For example, it has been observed that inhibition of MLCK does not lead to the complete dephosphorylation of MLC and that in MLCKdeficient mice, blood vessels remain responsive to vasoconstrictors. In addition, previous work has shown that the kinase IKK2 regulates migration and morphology in mouse embryonic fibroblasts, and that both of these processes depend on MLC phosphorylation. This led Ying and colleagues to ask whether IKK2 could be an MLC kinase in VSMCs. They found that not only was MLC phosphorylated by IKK2 in vitro and but also overexpression of IKK2 in VSMCs led to increased MLC phosphorylation. Conversely, inhibition of IKK2 decreased MLC phosphorylation and attenuated vasoconstrictive responses of isolated rat aortas. Furthermore, mice that lacked IKK2 in their VSMCs showed a decreased hypertensive response to three different vasoconstrictors. These results suggest that IKK2 inhibition could be a novel approach for decreasing hypertension.

The addition of three methyl groups to lysine 27 of histone H3—or H3K27me3 —is a repressive epigenetic modification that leads to gene silencing. The protein Jmjd3 is a H3K27 demethylase and therefore activates genes that have been suppressed by the modification. Recent evidence has shown that endothelial genes in adult pro-angiogenic cells are suppressed by H3K27me3. This led Ohtani and colleagues to investigate whether Jmjd3 might promote the expression of such genes earlier in development and thus might contribute to cardiovascular lineage commitment. To test the role of Jmjd3, the team created mouse embryonic stem cells that lacked Jmjd3 and examined their differentiation in vitro. They found that mesodermal, endodermal, and cardiomyoctye differentiation were all perturbed, as evinced by dramatically reduced expression of these lineage marker genes. The mesodermal marker genes, Brachyury and Mixl1 also showed an expected increase in H3K27me3 at their promoter regions. Mice generated from these embryonic stem cells died at early stages of embryo development, indicating an important role of this factor in the development of the cardiovascular system.

The calcium channel CaV1.2 plays an essential role in excitation-contraction coupling in cardiac muscle cells. Therefore, not surprisingly dysregulation of this channel has been implicated in electrical instabilities, arrhythmias, and even sudden cardiac death. The activity of CaV1.2 is controlled by β-adrenergic signaling, but the underlying mechanism remains incompletely understood. For example, it has been suggested that cleavage of CaV1.2 at alanine residue 1800 is required for β-adrenergic stimulation, and that this cleavage depends on a neighboring peptide motif. It has also been proposed that phosphorylation at serine residue 1700 is essential for β-adrenergic stimulation. But neither of these possibilities has been tested in native cardiomyocytes. Thus, Yang et al developed transgenic mice that expressed inducible, tissue-specific, and dihydropyridine-resistant versions of CaV1.2. Imparting dihydropyridine resistance enabled the investigators to separate transgenic CaV1.2 from the endogenous dihydropyridine-susceptible channel. The transgenic CaV1.2 carried mutations that disrupted either serine 1700 phosphorylation or the putative cleavage site residues. Yet surprisingly cleavage and β-adrenergic stimulation of CaV1.2 occurred as normal. These results highlight the importance of testing CaV1.2 in its native setting, and the new transgenic approach provides an ingenious and effective means to do so.

Stem cell therapy is a promising approach for promoting the repair of ischemic tissue. However, it is believed that, the proliferative and the reparative capacity of stem cells diminishes with aging. Therefore, finding ways to rejuvenate the flagging potential of stem cells could improve their therapeutic efficacy. Madonna and colleagues now suggest that transducing mesenchymal stem cells (MSCs) with two factors—telomerase reverse transcriptase (TERT) and myocardin—could achieve such rejuvenation and improve the myogenic potential of these stem cells. TERT is an enzyme that maintains the telomeres of chromosomes, which normally shorten with age, while myocardin regulates cardiovascular myogenic development. When both factors were expressed in MSCs via lentiviral expression plasmids, the cells exhibited improved proliferation, survival and myogenesis in vitro. Importantly, when injected into ischemic hind limb muscles of mice, the transduced cells improved arteriogenesis and blood flow to a greater degree than that achieved with non-transduced MSCs. Taken together these results indicate that overexpression of TERT and myocardin could improve the therapeutic potential of MSCs and their efficacy in treating vascular disorders such as myocardial ischemia and peripheral artery disease.

Proper differentiation of vascular smooth muscle cells (VSMCs) is not only essential for building blood vessels in the embryo, but also in adulthood, where abnormal VSMC differentiation is implicated in atherosclerosis, hypertension, and restenosis after angioplasty. Interestingly, VSMCs derive from 8 different progenitor cell types and exhibit differing characteristics depending on their origins. Xie and colleagues studied the neural crest cell (NCC) derived VSMCs, which ultimately locate to the aortic arch arteries, ascending aorta and carotid arteries. In vitro studies indicate that growth factor TGFβ regulates differentiation of NCC to VSMC, but in vivo experiments have given contrasting results. Xie and colleagues thus turned to a downstream target of TGFβ—Smad2—to resolve the issue. They deleted Smad2 from NCCs in mice and found that while NCCs still migrated to the aortic arch, they failed to differentiate to VSMCs. The VSMC layer in carotid arteries was also diminished. The team also showed that, in a mouse neural crest cell line, Smad2 interacted with transcription factor MRTFB—an important regulator of NCC to VSMC differentiation—which led to upregulation of VSMC markers. The authors therefore suggest that TGFβ signaling, via Smad2, is indeed important for NCC to VSMC differentiation.

After a heart attack, macrophages gather at the injury site to gobble-up dying cardiomyocytes. This feasting on cellular corpses, or efferocytosis, is essential for efficient resolution of inflammation, which in turn limits the degree of adverse cardiac remodeling and the risk of progression to heart failure. Indeed, it has been shown that defective cardiomyocyte clearance in older mice is associated with impaired heart repair, and that macrophages from older animals exhibit reduced efferocytosis. To learn more, Wan and colleagues examined macrophages with deletions of known efferocytosis receptors. They found only one—MERTK—that was essential for removal of necrotic cardiomyocytes through phagocytosis. They also found that mice lacking MERTK still recruited macrophages to their hearts following myocardial infarction. However, inflammation was slow to resolve, there were more dying and dead cardiomyocytes, infarct size was larger, and heart function was worse. Interestingly, while levels of the MERTK receptor increased in the hearts of wild-type mice 3 to 7 days after a myocardial infarction, the authors also found that some of the MERTK was cleaved into an inactive form. Why this inactivation occurs, whether it worsens with age, and whether it can be prevented to improve recovery after myocardial infarction are all avenues the authors plan to investigate.

Ludewig et al identify an inflammation inhibitor in mice that, after a stroke, limits the breakdown of the blood brain barrier and thus further injury.

After the initial hypoxic insult of an ischemic stroke, the problems are far from over. The resulting tissue damage leads to recruitment of immune cells, which cause inflammation with possible deterioration of the blood brain barrier and further brain damage. Immune cells express adhesion molecules that allow the cells to stick at the injury site and infiltrate the tissue. Blocking certain adhesion proteins has been shown to reduce cerebral damage after stroke in mice. But Ludewig and colleagues hypothesized that blocking the adhesion molecule CEACAM1 might have the opposite effect—because although it is also expressed on immune cells, it suppresses inflammation. They were right: Mice that lacked CEACAM1 faired worse after experimentally induced stroke. Their infarct sizes were larger, they had more pronounced cerebral inflammation, and their motility and neurological performance were more impaired. It turned out that without the anti-inflammatory action of CEACAM1, more immune cells that produced MMP-9—a secreted protease that promoted the breakdown of the blood brain barrier—were recruited to the ischemic brain. Together the results suggest that boosting CEACAM1 or inhibiting MMP-9 might be attractive ways to limit brain damage after stroke.

Atherosclerosis is a complex disease involving both environmental factors—for example, diet—and genes. Genetic loci that confer increased or decreased susceptibility to atherosclerosis have been identified in mice and man, but pinpointing the causative sequence changes at these loci is not easy. Out of the 44 or so modifier loci identified in mice, for example, the modifier sequences have been identified at just a handful. Now Rodriguez and colleagues add another locus to that list—Ath11. This locus, which associates with more severe atherosclerosis, maps to a particular stretch of mouse chromosome 10. The team identified five possible candidate genes in the region and analyzed their expression in atherosclerosis-prone mice that were either homozygous or heterozygous for the Ath11 modifier—and that had more or less severe atherosclerosis as a result. One gene, Raet1e, exhibited lower expression levels in the homozygotes. And reinstating high expression of Raet1e, by means of a transgene, reduced the severity of atherosclerosis. The team went on to identify the sequence change in the promoter of Raet1e responsible for the reduced expression. It is not clear why low levels of Raet1e worsen atherosclerosis, but the protein is known to interact with immune cells suggesting that an aberrant immune response may be to blame.

Quaking (QKI) is an RNA binding protein that regulates splicing, export and stability of messenger RNAs, and has been shown to be important for vascular development in the embryo. Van der Veer and colleagues investigated whether QKI might also have a role in the adult vasculature. They found that in healthy human vessels, smooth muscle cells (VSMCs) expressed almost undetectable levels of QKI, but that in restenotic vessels—where the VSMCs dedifferentiate and proliferate rapidly—the cells showed a dramatic increase in QKI expression. In concordance, mice that expressed a reduced amount of QKI exhibited less severe restenosis after vessel injury. VSMCs derived from these mice also displayed reduced proliferation, migration and extracellular matrix production—indicators of restenosis. The team went on to show that QKI interacted with the pre-mRNA of myocardin—an important promoter of VSMC maturation—and promoted the excision of one of myocardin’s exons. This splicing event generated a version of myocardin that increased VSMC proliferation. The authors therefore suggest that interfering with QKI activity could be a therapeutic path toward ameliorating pathogenic restenosis after vessel injury.

Recent research into arteriogenesis—the development of arteries and smaller arterioles—indicates that an intracellular signaling protein called synectin is involved. For example, mice and zebrafish that lack synectin exhibit impaired arterial morphogenesis. Their vessels are smaller sized with reduced diameters as well as reduced numbers of branches. Moraes et al were interested in which cell type might be responsible for these synectin-associated arterial defects. They generated mice that specifically lacked synectin in each of the two major artery cell types: vascular smooth muscle cells (VSMCs) and endothelial cells. They found that mice lacking synectin in endothelial cells but not those lacking synectin in their VSMCs exhibited virtually the same developmental arterial defects as the whole-mouse synectin deletion. Furthermore, when the researchers induced ischemia in the hind-limbs of adult mice, those lacking synectin in their endothelial cells had impaired recovery of blood flow while those lacking synectin in their VSMCs did not. The work provides a greater understanding of the molecular and cellular mechanisms behind artery formation, which will aid in the design of future therapies for ischemic vascular diseases, say the authors.

Fully differentiated vascular smooth muscle cells (VSMCs) in situ are generally non-proliferative, but after a vessel injury these cells dedifferentiate, proliferate and migrate. It is believed that such phenotype switching is an essential part of the healing process, but it can also lead to vessel narrowing, restenosis and other associated problems. Among the factors controlling this phenotype switch are microRNAs (miRs)—that suppress the expression of target mRNAs. While a number of such miRs have been identified in animal VSMCs, little is known about miRs in human VSMCs. Li and colleagues thus analyzed changes in miR expression in human VSMCs following induction of the phenotype switch. They found that miR-663 in particular, was dramatically downregulated. They also found that overexpression of miR-663 in VSMCs induced the expression of differentiation markers, whereas blocking miR-663 expression increased both proliferation and migration. MiR-663 targeted the mRNA of transcription factor JunB, suppressing its expression in human VSMCs. Although miR-663 is not conserved between humans and mice, its target sequence in the JunB mRNA is, and the team found that transfection of miR-663 into mice could suppress JunB expression and could block vessel narrowing. Boosting miR-663 activity or suppressing JunB might thus be strategies for preventing restenosis.

Schlueter and Brand investigate the origins of the heart’s proepicardium and discover that some of the cells derive from somatic mesoderm.

During embryogenesis, the proepicardium gives rise to epicardial cells as well as cells of the coronary vasculature and cardiac fibroblasts. But the origin of the proepicardium itself is unclear. In addition, it is also unclear how the proepicardium develops its characteristic left-right asymmetry—though the growth factor FGF8 and transcription factor SNAI1 are both known to be involved. Schlueter and Brand now show that a known target gene of SNAI1, a transcription factor called TWIST1, is asymmetrically expressed in lateral plate mesoderm of the early chick embryo, being highly expressed on the right-hand side. Furthermore, the team showed that these cells give rise to a population of proepicardial cells. They found that loss of TWIST1 expression in the right-hand mesoderm led to malformation of the proepicardium, while overexpression of TWIST in the left-hand mesoderm caused these cells to adopt characteristics of the right-hand proepicardium. The findings could have implications not only for understanding cardiac development, but also for understanding disease processes like myocardial fibrosis and for developing approaches for regenerative therapies.

Abonnenc et al confirm the role of microRNAs in extracellular matrix secretion by cardiac fibroblasts.

Changes to the composition of the extracellular matrix (ECM) contribute to the development of fibrosis, a characteristic feature of myocardial remodeling in a number of cardiac diseases. Previous studies have identified several microRNAs (miRs) that regulate cardiac fibrosis, including miR-30c and miR-29b. But a systematic analysis of the targets of these miRs is lacking. Rather than searching for messenger RNA targets of miR-30c and 29b—via computer prediction software or microarray analyses—Abonnenc and colleagues opted to search for proteins secreted by cardiac fibroblasts in response to the two miRs, thus allowing for the identification of both direct and indirect targets. The team used mass spectrometry to study the secretomes of primary mouse cardiac fibroblasts in which the two miRs were either over-expressed or inhibited. While miR-29b suppressed the expression of ECM and fibrosis proteins, including collagens, matrix metalloproteases, leukemia inhibitory factor (LIF) and insulin-like growth factor-1 (IGF-1), miR-30c had little effect on these proteins. The proteomic approach identified previously predicted and novel targets for the two miRs and, say the authors, offers a valuable resource for future studies into cardiac fibrosis.

During an intraplaque hemorrhage in an unstable atherosclerotic plaque, red blood cells rush in and accumulate. Once inside, they become prone to rapid lysis, resulting in the release of free iron molecules, which can oxidize lipids, induce cell death and ultimately promote atherosclerogenesis. But macrophages present in the plaque help to prevent such disastrous outcomes. Bories and colleagues wanted to identify which macrophages were involved in minimizing the damage. Hence, they compared two key macrophage populations—the M2 and resting macrophages (RM). They found that the M2 macrophages were more adept at ingesting red blood cells than RMs. As a result, M2 macrophages exhibited stronger—in fact almost exclusive—staining for iron content. The team went on to show that the uptake of iron by M2 macrophages induced the transcription factor LXR, which in turn promoted the expression of ferroportin, a protein involved in iron export. The iron released from macrophages by ferroportin is normally bound to a protein called transferrin, which renders the iron incapable of inducing oxidative damage. The authors suggest that boosting the activity of M2 macrophages or LXR could limit the progression of atherosclerotic lesions.

While angiogenesis is known to be problematic in several diseases, including cancer, it is essential for embryo development and for tissue growth and wound healing in adults. Angiogenesis is regulated by vascular endothelial growth factor (VEGF), which transforms endothelial cells of an existing vessel into tip cells that can then grow into new stalks. During this process, the protein DLL4 is activated in the tip cells and signals, via Notch, to suppress the expression of VEGF receptors in the cells of the growing stalk. This repression prevents the generation of too many tip cells. Indeed, loss of the DLL4-Notch signal results in formation of hyper-dense vascular networks. Adam and colleagues looked for proteins with the potential to modify angiogenesis by interacting with DLL4. They found SYNJ2BP. The protein was expressed in vascular endothelial cells and, when repressed, caused excessive microvascular density in an in vivo assay. Conversely, over-expression of SYNJ2BP prevented angiogenesis. The team showed that SYNJ2BP actually stabilizes the DLL4 protein, thus maintaining its Notch-inducing activity in the newer stalk cells. Enhancing the activity of SYNJ2BP or the DLL4-Notch pathway could therefore be a viable strategy for suppressing angiogenesis.

Autophagy, a catabolic process in which the cell digests its own components, might sound a bit macabre but it is an essential for survival during periods of energy starvation such as myocardial ischemia. By devouring and then digesting dysfunctional proteins and organelles, autophagy provides energy-poor cells with the substrates needed for ATP production, thus limiting the extent of tissue damage. The mechanisms controlling the induction and the progression of autophagy are not fully understood. It is known, however, that energy deprivation promotes the production of reactive oxygen species (ROS) in cells. Thus, Sciarretta and colleagues asked whether Nox4—an enzyme that generates ROS—is induced in energy-deprived cardiomyocytes and, if so, whether it could then regulate autophagy. The team showed that depriving cardiomyocytes of glucose, which is known to stimulate autophagy, led to increases in both Nox4 and ROS. Moreover, autophagy was suppressed and the cells died more readily when Nox4 was absent. In vivo studies revealed that the hearts of mice starved for 48 hours had increased levels of Nox4 and autophagy and that these increases were crucial for maintaining cardiac function. In fact, upon experimental ischemia, Nox4-null mice exhibited larger infarct sizes. Taken together, these results suggest that boosting Nox4 and autophagy in patients experiencing prolonged myocardial ischemia could protect against the loss of cardiac tissue.

Lipoprotein apheresis is a blood cleaning treatment in which the blood of patients with hypercholesterolemia is purged of the LDL-containing fraction. It is a highly effective method of removing LDL—often resulting in a decrease of up to 80%. Now Tavori et al report that an added benefit of this process (apheresis) is the removal of a protein called PCSK9. PCSK9 binds and suppresses the LDL-receptor, ultimately hindering the removal of LDL from blood. But recent work revealed that PSCK9 does not just bind the LDL receptor. It also binds LDL itself. Building on these observations, Tavori and colleagues hypothesized that along with LDL, lipoprotein apheresis might also remove PSCK9 from the blood—and indeed it did. In fact, apheresis removed much more PCSK9 than expected and much of it was LDL-free. The authors suggest that the removal of PSCK9 during apheresis could contribute to the maintenance of low levels of LDL between treatment sessions. Importantly, these findings lend further support to the development of PSCK9-inhibition strategies for use either alone, or in conjunction with apheresis.

Adult vascular endothelial cells are normally quiescent; however, after injury they proliferate, migrate, and contribute to the repair of existing vessels and/or the growth of new vessels. The growth factor VEGF, a major regulator of blood vessel growth, has been shown to activate several microRNAs (miRs)—small noncoding regulatory RNAs—in cultured endothelial cells. But Shi and colleagues were interested in finding such angiogenesis-related miRs expressed in native endothelium. The team therefore compared the miRs expressed in freshly isolated cells with those expressed in proliferating, cultured endothelial cells. They found that one specific microRNA, miR-223, was highly expressed in the freshly isolated cells, but was dramatically down-regulated once the cells were cultured. They also found that miR-223 was further suppressed by VEGF treatment, and overexpressing miR-223 attenuated VEGF-induced proliferation of endothelial cells. Furthermore, in vivo suppression of miR-223 boosted neovascularization after ischemia. The team went on to show that miR-223 exerts its antiangiogenic effects by directly suppressing the expression of β1 integrin—a known proangiogenesis factor. Thus it seems that miR-223 could be a new regulator of blood vessel growth and repair.

Although elevated blood levels of high-density lipoproteins (HDL) are associated with a decreased risk for heart disease, simply raising HDL in patients that have suffered acute cardiovascular events does not reduce the risk of a recurrence. Indeed, evidence suggests that HDL function is also impaired in patients with cardiovascular disease. Adams and colleagues thus compared the function of HDL isolated from healthy people with that from patients with chronic heart failure. While HDL is known to protect the vascular endothelium in a number of ways - by promoting reverse cholesterol transport, diminishing inflammation, and stimulating vasodilation via nitric oxide (NO) production, the team focused on this latter effect only. They found that, compared with HDL from healthy subjects, HDL from patients with heart failure had a reduced capacity to stimulate NO production in cultured human endothelial cells. They then tested HDL isolated from subjects that had undergone 12 weeks of aerobic training. Their results showed that exercise training enhanced the NO producing capacity of HDL in heart failure patients, although in healthy subjects, exercise training had no effect on HDL. These findings suggest that heart failure impairs HDL function and that improving HDL function may be one of the reasons why exercise training benefits these patients.

Footnotes

Circulation Research “In This Issue” Anthology: In the May 14, 2010 issue, we inaugurated a new feature, the In This Issue page, which highlights articles that the editors feel are particularly important by providing a short synopsis written in a language that can be understood by the non-specialist. Our purpose in creating this new feature was two-fold: first, to call the attention of readers to important articles and, second, to enable all readers to grasp, easily and quickly, the significance of these papers. We believe that this initiative has been successful and, as a further service to our readers, provide below a compendium of these pages from the previous year, 2013. Each summary is accompanied by the corresponding citation, so that readers can easily find the entire article should they wish to read it. This remarkable anthology of outstanding articles attests to the diversity and depth of the work published in Circulation Research. — The Editors

. Induction of vascular insulin resistance and endothelin-1 expression and acceleration of atherosclerosis by the overexpression of protein kinase C-β isoform in the endothelium.Circ Res. 2013;113:418–427.

. Transplantation of mesenchymal cells rejuvenated by the overexpression of telomerase and myocardin promotes revascularization and tissue repair in a murine model of hindlimb ischemia.Circ Res. 2013;113:902–914.